VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center in 2023.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) is a Bioinformatics Resource Center funded by the National Institutes of Health with additional funding from the Wellcome Trust. VEuPathDB supports >600 organisms that comprise invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Since 2004, VEuPathDB has analyzed omics data from the public domain using contemporary bioinformatic workflows, including orthology predictions via OrthoMCL, and integrated the analysis results with analysis tools, visualizations, and advanced search capabilities. The unique data mining platform coupled with >3000 pre-analyzed data sets facilitates the exploration of pertinent omics data in support of hypothesis driven research. Comparisons are easily made across data sets, data types and organisms. A Galaxy workspace offers the opportunity for the analysis of private large-scale datasets and for porting to VEuPathDB for comparisons with integrated data. The MapVEu tool provides a platform for exploration of spatially resolved data such as vector surveillance and insecticide resistance monitoring. To address the growing body of omics data and advances in laboratory techniques, VEuPathDB has added several new data types, searches and features, improved the Galaxy workspace environment, redesigned the MapVEu interface and updated the infrastructure to accommodate these changes.

Intracellular Plasmodium aquaporin 2 is important for sporozoite production in the mosquito vector and malaria transmission.

Malaria remains a devastating disease and, with current measures failing to control its transmission, there is a need for novel interventions. A family of proteins that have long been pursued as potential intervention targets are aquaporins, which are channels facilitating the movement of water and other solutes across membranes. We identify an aquaporin in malaria parasites and demonstrate that it is important for completion of Plasmodium development in the mosquito vector. Disruption of AQP2 in the human parasite Plasmodium falciparum and the rodent parasite Plasmodium berghei blocks sporozoite production inside oocysts established on mosquito midguts, greatly limiting parasite infection of salivary glands and transmission to a new host. In vivo epitope tagging of AQP2 in P. berghei, combined with immunofluorescence assays, reveals that the protein is localized in vesicle-like organelles found in the cytoplasm of gametocytes, ookinetes, and sporozoites. The number of these organelles varies between individual parasites and lifecycle stages suggesting that they are likely part of a dynamic endomembrane system. Phylogenetic analysis confirms that AQP2 is unique to malaria and closely related parasites and most closely resembles intracellular aquaporins. Structure prediction analyses identify several unusual features, including a large accessory extracellular loop and an arginine-to-phenylalanine substitution in the selectivity filter principally determining pore function, a unique feature among known aquaporins. This in conjunction with the importance of AQP2 for malaria transmission suggests that AQP2 may be a fruitful target of antimalarial interventions.

Testing non-autonomous antimalarial gene drive effectors using self-eliminating drivers in the African mosquito vector Anopheles gambiae.

Gene drives for mosquito population modification are novel tools for malaria control. Strategies to safely test antimalarial effectors in the field are required. Here, we modified the Anopheles gambiae zpg locus to host a CRISPR/Cas9 integral gene drive allele (zpgD) and characterized its behaviour and resistance profile. We found that zpgD dominantly sterilizes females but can induce efficient drive at other loci when it itself encounters resistance. We combined zpgD with multiple previously characterized non-autonomous payload drives and found that, as zpgD self-eliminates, it leads to conversion of mosquito cage populations at these loci. Our results demonstrate how self-eliminating drivers could allow safe testing of non-autonomous effector-traits by local population modification. They also suggest that after engendering resistance, gene drives intended for population suppression could nevertheless serve to propagate subsequently released non-autonomous payload genes, allowing modification of vector populations initially targeted for suppression.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

Anopheles coluzzii stearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding.

Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne diseases.

PIMMS43 is required for malaria parasite immune evasion and sporogonic development in the mosquito vector.

After being ingested by a female Anopheles mosquito during a bloodmeal on an infected host, and before they can reach the mosquito salivary glands to be transmitted to a new host, Plasmodium parasites must establish an infection of the mosquito midgut in the form of oocysts. To achieve this, they must first survive a series of robust innate immune responses that take place prior to, during, and immediately after ookinete traversal of the midgut epithelium. Understanding how parasites may evade these responses could highlight new ways to block malaria transmission. We show that an ookinete and sporozoite surface protein designated as PIMMS43 (Plasmodium Infection of the Mosquito Midgut Screen 43) is required for parasite evasion of the Anopheles coluzzii complement-like response. Disruption of PIMMS43 in the rodent malaria parasite Plasmodium berghei triggers robust complement activation and ookinete elimination upon mosquito midgut traversal. Silencing components of the complement-like system through RNAi largely restores ookinete-to-oocyst transition but oocysts remain small in size and produce a very small number of sporozoites that additionally are not infectious, indicating that PIMMS43 is also essential for sporogonic development in the oocyst. Antibodies that bind PIMMS43 interfere with parasite immune evasion when ingested with the infectious blood meal and significantly reduce the prevalence and intensity of infection. PIMMS43 genetic structure across African Plasmodium falciparum populations indicates allelic adaptation to sympatric vector populations. These data add to our understanding of mosquito-parasite interactions and identify PIMMS43 as a target of malaria transmission blocking.

Unravelling population structure heterogeneity within the genome of the malaria vector Anopheles gambiae.

BACKGROUND: Whole genome re-sequencing provides powerful data for population genomic studies, allowing robust inferences of population structure, gene flow and evolutionary history. For the major malaria vector in Africa, Anopheles gambiae, other genetic aspects such as selection and adaptation are also important. In the present study, we explore population genetic variation from genome-wide sequencing of 765 An. gambiae and An. coluzzii specimens collected from across Africa. We used t-SNE, a recently popularized dimensionality reduction method, to create a 2D-map of An. gambiae and An. coluzzii genes that reflect their population structure similarities. RESULTS: The map allows intuitive navigation among genes distributed throughout the so-called "mainland" and numerous surrounding "island-like" gene clusters. These gene clusters of various sizes correspond predominantly to low recombination genomic regions such as inversions and centromeres, and also to recent selective sweeps. Because this mosquito species complex has been studied extensively, we were able to support our interpretations with previously published findings. Several novel observations and hypotheses are also made, including selective sweeps and a multi-locus selection event in Guinea-Bissau, a known intense hybridization zone between An. gambiae and An. coluzzii. CONCLUSIONS: Our results present a rich dataset that could be utilized in functional investigations aiming to shed light onto An. gambiae s.l genome evolution and eventual speciation. In addition, the methodology presented here can be used to further characterize other species not so well studied as An. gambiae, shortening the time required to progress from field sampling to the identification of genes and genomic regions under unique evolutionary processes.

Anopheles arabiensis hotspots along intermittent rivers drive malaria dynamics in semi-arid areas of Central Ethiopia.

BACKGROUND: Understanding malaria vector's population dynamics and their spatial distribution is important to define when and where the largest infection risks occur and implement appropriate control strategies. In this study, the seasonal spatio-temporal dynamics of the malaria vector population and transmission intensity along intermittent rivers in a semi-arid area of central Ethiopia were investigated. METHODS: Mosquitoes were collected monthly from five clusters, 2 close to a river and 3 away from a river, using pyrethrum spray catches from November 2014 to July 2016. Mosquito abundance was analysed by the mixed Poisson regression model. The human blood index and sporozoite rate was compared between seasons by a logistic regression model. RESULTS: A total of 2784 adult female Anopheles gambiae sensu lato (s.l.) were collected during the data collection period. All tested mosquitoes (n = 696) were identified as Anopheles arabiensis by polymerase chain reaction. The average daily household count was significantly higher (P = 0.037) in the clusters close to the river at 5.35 (95% CI 2.41-11.85) compared to the clusters away from the river at 0.033 (95% CI 0.02-0.05). Comparing the effect of vicinity of the river by season, a significant effect of closeness to the river was found during the dry season (P = 0.027) and transition from dry to wet season (P = 0.032). Overall, An. arabiensis had higher bovine blood index (62.8%) as compared to human blood index (23.8%), ovine blood index (9.2%) and canine blood index (0.1%). The overall sporozoite rate was 3.9% and 0% for clusters close to and away from the river, respectively. The overall Plasmodium falciparum and Plasmodium vivax entomologic inoculation rates for An. arabiensis in clusters close to the river were 0.8 and 2.2 infective bites per person/year, respectively. CONCLUSION: Mosquito abundance and malaria transmission intensity in clusters close to the river were higher which could be attributed to the riverine breeding sites. Thus, vector control interventions including targeted larval source management should be implemented to reduce the risk of malaria infection in the area.

Microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes.

Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector Anopheles coluzzii. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of Enterobacteriaceae within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract.

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity.

BACKGROUND: The development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive. METHODS: The SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed. RESULTS: A detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector. CONCLUSION: A robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

The serine protease homolog CLIPA14 modulates the intensity of the immune response in the mosquito Anopheles gambiae.

Clip domain serine protease homologs (SPHs) are positive and negative regulators of Anopheles gambiae immune responses mediated by the complement-like protein TEP1 against Plasmodium malaria parasites and other microbial infections. We have previously reported that the SPH CLIPA2 is a negative regulator of the TEP1-mediated response by showing that CLIPA2 knockdown (kd) enhances mosquito resistance to infections with fungi, bacteria, and Plasmodium parasites. Here, we identify another SPH, CLIPA14, as a novel regulator of mosquito immunity. We found that CLIPA14 is a hemolymph protein that is rapidly cleaved following a systemic infection. CLIPA14 kd mosquitoes elicited a potent melanization response against Plasmodium berghei ookinetes and exhibited significantly increased resistance to Plasmodium infections as well as to systemic and oral bacterial infections. The activity of the enzyme phenoloxidase, which initiates melanin biosynthesis, dramatically increased in the hemolymph of CLIPA14 kd mosquitoes in response to systemic bacterial infections. Ookinete melanization and hemolymph phenoloxidase activity were further increased after cosilencing CLIPA14 and CLIPA2, suggesting that these two SPHs act in concert to control the melanization response. Interestingly, CLIPA14 RNAi phenotypes and its infection-induced cleavage were abolished in a TEP1 loss-of-function background. Our results suggest that a complex network of SPHs functions downstream of TEP1 to regulate the melanization reaction.

Plasmodium berghei PIMMS2 Promotes Ookinete Invasion of the Anopheles gambiae Mosquito Midgut.

Mosquito midgut stages of the malaria parasite present an attractive biological system to study host-parasite interactions and develop interventions to block disease transmission. Mosquito infection ensues upon oocyst development that follows ookinete invasion and traversal of the mosquito midgut epithelium. Here, we report the characterization of PIMMS2 (Plasmodium invasion of mosquito midgut screen candidate 2), a Plasmodium berghei protein with structural similarities to subtilisin-like proteins. PIMMS2 orthologs are present in the genomes of all plasmodia and are mapped between the subtilisin-encoding genes SUB1 and SUB3 P. berghei PIMMS2 is specifically expressed in zygotes and ookinetes and is localized on the ookinete surface. Loss of PIMMS2 function through gene disruption by homologous recombination leads to normal development of motile ookinetes that exhibit a severely impaired capacity to traverse the mosquito midgut and transform to oocysts. Genetic complementation of the disrupted locus with a mutated PIMMS2 allele reveals that amino acid residues corresponding to the putative subtilisin-like catalytic triad are important but not essential for protein function. Our data demonstrate that PIMMS2 is a novel ookinete-specific protein that promotes parasite traversal of the mosquito midgut epithelium and establishment of mosquito infection.

Genetic dissection of Anopheles gambiae gut epithelial responses to Serratia marcescens.

Genetic variation in the mosquito Anopheles gambiae profoundly influences its ability to transmit malaria. Mosquito gut bacteria are shown to influence the outcome of infections with Plasmodium parasites and are also thought to exert a strong drive on genetic variation through natural selection; however, a link between antibacterial effects and genetic variation is yet to emerge. Here, we combined SNP genotyping and expression profiling with phenotypic analyses of candidate genes by RNAi-mediated silencing and 454 pyrosequencing to investigate this intricate biological system. We identified 138 An. gambiae genes to be genetically associated with the outcome of Serratia marcescens infection, including the peptidoglycan recognition receptor PGRPLC that triggers activation of the antibacterial IMD/REL2 pathway and the epidermal growth factor receptor EGFR. Silencing of three genes encoding type III fibronectin domain proteins (FN3Ds) increased the Serratia load and altered the gut microbiota composition in favor of Enterobacteriaceae. These data suggest that natural genetic variation in immune-related genes can shape the bacterial population structure of the mosquito gut with high specificity. Importantly, FN3D2 encodes a homolog of the hypervariable pattern recognition receptor Dscam, suggesting that pathogen-specific recognition may involve a broader family of immune factors. Additionally, we showed that silencing the gene encoding the gustatory receptor Gr9 that is also associated with the Serratia infection phenotype drastically increased Serratia levels. The Gr9 antibacterial activity appears to be related to mosquito feeding behavior and to mostly rely on changes of neuropeptide F expression, together suggesting a behavioral immune response following Serratia infection. Our findings reveal that the mosquito response to oral Serratia infection comprises both an epithelial and a behavioral immune component.

Transcriptional silencing and activation of paternal DNA during Plasmodium berghei zygotic development and transformation to oocyst.

The malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of Plasmodium zygotic development and ookinete-to-oocyst transition. To achieve this we used in vitro and in vivo cross-fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage-specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of Plasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.

Characterization of Plasmodium developmental transcriptomes in Anopheles gambiae midgut reveals novel regulators of malaria transmission.

The passage through the mosquito is a major bottleneck for malaria parasite populations and a target of interventions aiming to block disease transmission. Here, we used DNA microarrays to profile the developmental transcriptomes of the rodent malaria parasite Plasmodium berghei in vivo, in the midgut of Anopheles gambiae mosquitoes, from parasite stages in the midgut blood bolus to sporulating oocysts on the basal gut wall. Data analysis identified several distinct transcriptional programmes encompassing genes putatively involved in developmental processes or in interactions with the mosquito. At least two of these programmes are associated with the ookinete development that is linked to mosquito midgut invasion and establishment of infection. Targeted disruption by homologous recombination of two of these genes resulted in mutant parasites exhibiting notable infection phenotypes. GAMER encodes a short polypeptide with granular localization in the gametocyte cytoplasm and shows a highly penetrant loss-of-function phenotype manifested as greatly reduced ookinete numbers, linked to impaired male gamete release. HADO encodes a putative magnesium phosphatase with distinctive cortical localization along the concave ookinete periphery. Disruption of HADO compromises ookinete development leading to significant reduction of oocyst numbers. Our data provide important insights into the molecular framework underpinning Plasmodium development in the mosquito and identifies two genes with important functions at initial stages of parasite development in the mosquito midgut.

Comprehensive genetic dissection of the hemocyte immune response in the malaria mosquito Anopheles gambiae.

Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca²âº channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.

The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae.

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1(cut) that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals.

Adaptive divergence between incipient species of Anopheles gambiae increases resistance to Plasmodium.

The African malaria mosquito Anopheles gambiae is diversifying into ecotypes known as M and S forms. This process is thought to be promoted by adaptation to different larval habitats, but its genetic underpinnings remain elusive. To identify candidate targets of divergent natural selection in M and S, we performed genomewide scanning in paired population samples from Mali, followed by resequencing and genotyping from five locations in West, Central, and East Africa. Genome scans revealed a significant peak of M-S divergence on chromosome 3L, overlapping five known or suspected immune response genes. Resequencing implicated a selective target at or near the TEP1 gene, whose complement C3-like product has antiparasitic and antibacterial activity. Sequencing and allele-specific genotyping showed that an allelic variant of TEP1 has been swept to fixation in M samples from Mali and Burkina Faso and is spreading into neighboring Ghana, but is absent from M sampled in Cameroon, and from all sampled S populations. Sequence comparison demonstrates that this allele is related to, but distinct from, TEP1 alleles of known resistance phenotype. Experimental parasite infections of advanced mosquito intercrosses demonstrated a strong association between this TEP1 variant and resistance to both rodent malaria and the native human malaria parasite Plasmodium falciparum. Although malaria parasites may not be direct agents of pathogen-mediated selection at TEP1 in nature--where larvae may be the more vulnerable life stage--the process of adaptive divergence between M and S has potential consequences for malaria transmission.

Immune interactions between mosquitoes and microbes during midgut colonization.

Mosquitoes encounter diverse microbes during their lifetime, including symbiotic bacteria, shaping their midgut ecosystem. The organization of the midgut supports microbiota persistence while defending against potential pathogens. The influx of nutrients during blood feeding triggers bacterial proliferation, challenging host homeostasis. Immune responses, aimed at controlling bacterial overgrowth, impact blood-borne pathogens such as malaria parasites. However, parasites deploy evasion strategies against mosquito immunity. Leveraging these mechanisms could help engineer malaria-resistant mosquitoes, offering a transformative tool for malaria elimination.

Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1.

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.